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(11) | EP 1 323 826 A1 |
| (12) | EUROPEAN PATENT APPLICATION |
| published in accordance with Art. 158(3) EPC |
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| (54) | A CELL LINE EXPRESSING MUTATED HUMAN TISSUE - TYPE PLASMINOGEN ACTIVATOR, THE CONSTRUCTING STRATEGY THEREOF AND METHOD OF PREPARING EXPRESSED PROTEIN |
| (57) The invention involves a cell line which can express human mutated tissue-type plasminogen
activator (TNK-TPA), and its preparation methods. The collection No. of cell line
in this invention is CCTCC C200006. We firstly use a DNA fragment from the short arms
of human D, G group chromosomes or its homologous to construct a recombinant human
source gene vector-TNK-TPA (collection number is CCTC m200032); TNK-TPA gene is then
transferred into the target site of nucleolus organizing region (NOR) of human D,
G group chromosomes in host cell HT1080 by the recombinant and the cell line is attained
after screening, which can be used for manufacturing protein. |
SUMMARY OF THE INVENTION
[0001] The invention involves a cell line specifically expressing mutated human tissue-type plasminogen activator (TNK-TPA), cell line's construction strategy and its application in manufacturing recombinant gene medicine of human tissue-type plasminogen activator.
BACKGROUND OF THE INVENTION
[0002] Human tissue-type plasminogen activator(t-PA) can dissolve local thrombus in blood vessel and recanalize the blocked vessel by activating plasminogen. At present, American Genetech Company is the only one can develop recombinant human t-PA(rt-PA) at a large scale by means of gene project technology and has developed successfully rt-PA medicine. The first generation of rt-PA made in this company has become of choice treatment to thrombotic diseases, but it has some shortcomings. First, it's price is rather high with $1375 per vial on international market; second, its function has some weak points: 1)short half-life, just 3 to 5 minutes; 2)low specific affinity with fibrin of gore; 3) activity and efficacy should be further improved. Much more medicine dose should be given because of short half-life. Low specificity and activity give rise to two results: on one hand, there is still 10-20% patient's thrombus can't be completely dissolved by rt-PA, on the other hand, it can results in fibrinolysis over all the body and affects normal cruor mechanism so as to cause severe complications such as bleeding, especially in skull and digestive tract. In terms of the above reasons, some foreign companies have got down to develop the second generation of rt-PA since the -PA was used in the clinical. They modified the rt-PA gene in order to eliminate or weaken the above weak points. TNK-TPA is a kind of mutated form which is universally researched. For example, Keyt BA, Paoni NF. Refino rt CJ, et al proclaimed "a Faster-acting And More Potent Form of Tissue Plasminogen Activator". ((Proc Natl Acad Sci USA) 1997 Apr 26; 91(9):3670-4.). It was constructed on the basis of three sites of t-PA gene site-directed mutation. Two sites locate in Kringlel function domain, T103N and Q117N; one locates in Protease domain, KHRR296-299AAAA.T103N adds a glycosylation site which increases specificity of combination of TNK-tPA and fibrin and decreases its cleanup rate; Q117N destroys combination of Kl region and mannose and the action domain of KUPFFER cell in liver, and prolongs half-life; KHRR296-299AAAA destroys functional region of PAI-1 and prolongs its half-life, too. As for the development of TNK-TPA, it is universal to adopt ordinary eukaryotic gene expression vector. using CHO cell as host cell, TNK-TPA integrate randomly into the expression system of host cell genome. For example. Wu Benchuan, et al announced the above expression system and preparation method of expressed protein in the article "Purification and identification of recombinant tissue-type plasminogen activator" published in <Development of Biochemistry and Biophysics>( 1997,24 (1):71-75)
DETAILED DESCRIPTION OF THE INVENTION
[0003] The inventor discovered two families carrying an extra bi-satellite microchromosome (BM) in these two families with 2 and 3 generations, the BM does no harm to human body. The families show normal phenotype. It is reported 17 similar families in the world, and nobody conceived of assembling human gene vector using the small chromosome elements. The applicant put forward the structure. He found out that the chromosomes come from short arms of human D, G group chromosomes 13,14,15,21,22 by FISH technique. The short arms of human D, G group are nucleolus organizing region abundant in ribosome DNA (rDNA). These regions exist different polymorphisms with different length in human, and the genes in these regions can be actively transcribed in the cell metaphase. So he inferred that specific DNA fragments can be used as leading sequence if they were isolated from BM, and the gene of interest can be transferred into nucleolus organizing region in short arms of human D, G group chromosomes, the gene should be relatively highly, stably and harmlessly expressed. The following example can prove this strongly.
[0004] The inventor at first constructed specific pUC19 library of the microchromosomes by micro-dissection method and got single copy fragment by screening the library. The fragment proved by FISH technique comes from short arms of human D, G group chromosomes and the single copy was used as probe to screen human PAC genomic library. Then, a 120kb of BM specific DNA fragment which comes from BM and short arms of human D, G group chromosomes was confirmed by FISH technique (Fig. 1). Gene concerned important physiological function is not found by analyzing sequence of this BM specific fragment (BMSF). So it is safe to take it as target. The applicant further used a small fragment from BMSF as leading sequence to construct gene vector.
[0005] Based on above evidences, the applicant holds that a gene vector by using the DNA sequence without vital physiological function- related gene or a DNA sequence with identity to short arms of human D, G group chromosomes as the leading sequence can site-directly introduce target gene to the short arms of human D, G group chromosomes and the cell line for this invention can then be obtained.
[0006] Different vectors can be constructed on the basis of leading sequence according to the present technique, and then we can get recombinant vector-TNK-TPA by ligating TNK-TPA gene into the constructed vector. The construction methods of vector and recombinant vector-TNK-TPA are commonly used. Using the recombinant above, the target gene TNK-TPA can be also transferred into host cell by routine method.
[0007] The example of the invention gives in detail the identification of the 120kb specific DNA sequence from short arms of human D, G group chromosomes, the construction of the vector using a 3.8kb fragment from the 120kb BMSF as leading sequence. The full sequence of the vector is given in SEQ No 1. Then target gene is ligated into the vector and recombinant vector-TNK-TPA is achieved. The inserting site of TNK-TPA gene is at 5910 of the vector. The bacterial strain contained recombinant vector-TNK-TPA is preserved in the China Typical Culture Collection Center (Wuhan University, China. Wuhan 430072) on Sept. 29, 2000, collection number is CCTCCM200032. The preservator names it Escherichia coli JM 109/JH-S/pNS-TPA.
[0008] According to the way above, the invention got a novel cell line with target gene TNK-TPA integrated in short arms of D, G group chromosomes of HT1080 cell. The cell line was accepted by China Typical Culture Collection Center (Wuhan University, China. Wuhan 430072) on August 18, 2000, and preservation number is CCTCC C200006. The cell line is human fibrosarcoma cell line, which is human gene-transferred cell. The preservator names it human fibrosarcoma cell line /JH-2/TPA. The preserved is a pure cultivative cell line, the culture condition is 10% calf serum in DMEM , PH is 7.0-7.4 ,37°C.
[0010] The purified protein expressed by the cell line of the invention has been confirmed to be the target protein by Western blotting and amino acid sequence analysis.
[0011] The invention obtained a novel cell line by transferring site-directly recombinant human-source gene vector/TNK-TPA cDNA into D,G group chromosomes of human HT1080 cell and gave a method to produce TNK-TPA. Compared with the existing technology, it dramatically decreases cost of TNK-TPA because of its efficient and stable expression.
Figure 1 shows FISH mapping of 120kb fragment cloned in PAC;
Figure 2 is a circular map of the gene vector described in the invention (length of gene vector sequence: 11162bp); pGEM-7 (8267-11162):vector replication component and prokaryote screening system; TK(1-2840): eukaryote negative screen gene, using TK promoter and TK polyA signal; Neo(4342―5910):eukaryote positive screen gene, using sv40 promoter and sv40 polyA signal; GLS (2841―4341,5911―8267):leading sequence for gene targeting; Cloning site (5910): insert-site of target gene;
Figure 3 is FISH mapping of targeted TNK-TPA gene in positively transformed cell colony, this revealed that TNK-TPA gene has been site-directly transferred into short arms of D,G group chromosomes by the vector;
Figure 4 is Western blotting result of purified TNK-TPA protein, 1∼4 are purified TNK-TPA protein, "―"suggests negative control;
EXAMPLE ONE
Preparation of gene leading sequence in the invention
[0012]
1 Isolation of PAC clone with gene leading sequence
1.1 Construct BM specific pUC19 library through micro-dissection, PCR, microcloning (Deng H-X, Yoshirua K, Dirks RW, et al. Hum Genet 1992,89:13.)
1.2 Identification of BM specific single copy DNA
(1)preparation of colony matrix membrane: draw a 14×14 square wells on two nylon membranes, labeled as A and B, and put into two plates with solid LB, respectively, then pick white clone randomly from library plates and inoculate to the 14×12 square wells of two membranes with same coordinate, add 100ng single copy DNA to the 13th line as positive control, and then add 100ng gDNA to 14th line as negative control, two plates are incubated for 10∼12 hours at 37°C,and membrane B is stored at 4 °C, remove membrane A from plate, and treat on a filter paper soaked by following solution: 10%SDS, 5 minutes,0.5N NaOH/1.5M NaCl, 3 minutes, 1.5M NaCl/0.5M Tris.HCl, 3 minutes, 2×SSC/0.2M Tris.HCl, 10 minutes, 80 °C vacuum dry 2 hours, keep for use.
(2)Preparation of gDNA probe
Sample 50∼75ng gDNA, add asepsis water to 11ml, boil and denature at 100 °C for 10 minutes, the following system is used as random primer labeling reaction:
| 2mM dNTP(dATP-) | 3µl |
| primer mixture | 2µl |
| Klenow enzyme | 1µl |
| α-32p-dATP | 3µl |
(3)Hybridization: colony matrix membrane is immersed in 2 × SSC 10 minutes, wipe out lightly colony fragments on membrane surface, 65°C, pre-hybridize in 5ml hybridization solution over 30 minutes. Sample probe solution to boil and denature 10minutes at 100 °C according to the concentration of 1.2×106cpm/ml and measure liquid scintillation value, add 5ml fresh hybridization solution , hybridize with colony lattice membrane over 12 hours at 65°C,wash membrane according to following conditions: 2×SSC/0.1%SDS, 10 minutes at room temperature, 2× SSC/0.1%SDS, 10 minutes at 65°C,0.1%×SSC/0.1%SDS,10 minutes at 65°C, -70°C, autoradiograph, none or weak hybridization signal is primarily regarded as single copy.
(4)Sequence analysis, Southern blotting: pick clone without hybridization signal on the corresponding position of membrane B, culture it in small scale, sequence plasmid DNA extracted, the sequence of single copy DNA shows no similarity compared with the database of GenBank. At last, digest the plasmid by EcoR I enzyme and isolate the insert DNA by random primer method, label the insert by α-32p-dATP and hybridize with gDNA digested by EcoRI on a piece of nylon membrane, those showing 1 or 2 hybridization bands are single copy DNA clones.
1.3 Identification of PAC clone with BM specific sequence and short arms of human D, G group chromosomes
(1) Screen human PAC gDNA and acquire positive clone
(2) Label 260bp single copy probe P8-7 with α-32p-dATP by random primer method→purify probe by G-50 column (medium granularity) →ready for use 4°C →immerse 7 PAC membranes in 2×SSC 10 minutes→pre-hybridize 3 hours,55°C →denature probe 10 minutes, 100°C→add to 50ml hybridization solution purchased commercially to a final concentration of 4.6×105cpm/ml and hybridize with PAC membrane 1 hour 65°C→wash membrane: 2×SSC ,once a time 10 minutes at room temperature, 2 × SSC/0.1%SDS,10 minutes at 65°C twice→apply to X-ray film, autoradiograph at 70°C 12 hours→develop X-ray film→read the positive clones number following instruction.
(2) Pick randomly positive clones and number from five different plates, purchase PAC clone.
1.4 Hybridization of the DNA fragment in PAC clone with metaphase cell confirm that the insert DNA in positive PAC clone originates from short arms of human D, G group chromosomes through FISH, as the figure 1 shows.
The above experimental methods refer to the Molecular Cloning written by J Sambrook et al., second Edition, Cold Spring Harbor laboratory Press,1989.
2. Isolation of the leading sequence.
Main material: β-agarase(Bio-Labs), Not I Agarose
(1) Digest the plasmid PAC169 by Not I enzyme
(2) Isolate an around 120kb insert DNA through PFGE.
Pulse electrophoresis conditions: electrophoresis buffer: 0.5×TBE High strength Analytical
Grade Agarose: (Bio-Rad, low Melting point agarose LMP)1%,
Switch time: from two to fifteen second,
electrophoresis time:18h.voltage: 6v/cm,angle:120° ,temperature: 14 °C.
(3) After electrophoresis, stain the gel with EB (0.2ug/ml) thirty minutes. According to the Marker display, cut that 120kb DNA band with sterile knife.
(4) Treat the gel cut out by β-agarase and precipitate with enthanol.
EXAMPLE 2
Preparation of gene vector in the invention:
[0013]
1. The construction of gene vector and induction of target gene
1.1 Construct gene vector
1.1.1 Nsi I and Stu I (blunt enzyme) digest PAC DNA, run the sample on a common agarose gel, recover the 3.8kb DNA and purify the DNA by electric elution.
1.1.2 pGEM-TK vector DNA is digested with Hind III, and then digested with Klenow further to generate the blunt ends.
1.1.3 The blunt ends of pGEM-TK/Hind III is further digested with Nsi I.
1.1.4 The purified 3.8kb/Nsi I+Stu I digested DNA and the Nsi I digested pGEM-TK are ligated for 17 hours at 16°C.
1.1.5 The ligated product is transformed into JM109 competent bacteria, culture 18 hours at 37°C in an ampicillin plate.
1.1.6 Pick randomly monoclones and identify the positive clones with Nsi I and Nhe I double digestion.
1.1.7 The plasmid pCDN-GPR is digested with Xba I and Nhe I to acquire the Xbal+Nhel digested Neo gene.
1.1.8 Construct pNS2 gene vector by ligating the Xbal+Nhel digested Neo gene with the pGEM-TK-3.8kb/Nhe I fragment.
2.1 Induction of TNK-TPA gene
2.1.1 Clone TNK-TPA (CDS) into pcDNA3.1(-).
2.1.2 Design the primers TPCF and TPCR to amplify TNK-TPA gene and expression element
(CMV promoter and BGH polyA signal), Avr II restrictive sites are added to the two
ends of the primers.
2.1.3 Digest TNK-TPA gene and expression element (CMV promoter and BGH polyA signal) with AvrII, ligate it to the pNS2 vector digested with Nhe I.
2.1.4 Transform the ligated product into recombinant JM109 E.coci and gain bacterial strain containing vector-TNK-TPA (collection number is CCTCC M200032).
The above experimental methods refer to the Molecular Cloning written by .J Sambrook et al., second edition, Cold Spring Harbor laboratory Press.1989.
3 The gene vector extraction.
3.1 Material:
3.1.1 QIAGE Plasmid Maxi Kit
3.1.2 Culture medium: liquid LB
| Trypton | 5g |
| Yeast extract | 2.5g |
| NaCI | 2.5g |
| Add H2O to 500ml | |
| Sterilize by autoclaving | |
3.1.3 Ampicillin: 100mg/ml(1000×)
3.2 Method:
1) Pick positive monoclones and inoculate them in 3ml LB (Amp+), incubate for 1 h at 37°C, 250rpm.
2) Inoculate 100ul of primary culture in 100 ml LB (Amp+), incubate for 16 h at 37 °C,250rpm.
3) Harvest the bacteria by centrifugation at 6000×g for 15 minutes at 4°C
4) Resuspend the bacterial pellet in 10ml of buffer P1.
5) Add 10ml of buffer P2, mix gently but thoroughly 6 times, and incubate at room temperature for 5 minutes.
6) Add 10ml of chilled buffer P3, mix gently by inverting 6 times, and incubate on ice for 20 minutes.
7) Centrifuge at 2000× g for 30 min at 4°C.
8) Transfer supernatant containing plasmid DNA promptly into a 40ml high-speed centrifuge tube and centrifuge at 2000× g for 15 min at 4°C.
9) Equilibrate a QIGEN tip 500 by applying 10ml buffer QBT.
10) Transfer the supernatant to the QIGAE-tip 500 and allow it to pass through this column.
11) Wash the Q1GAE-tip with 30ml buffer QC.
12) Elute DNA with 15ml QF and collect elution.
13) Precipitate DNA by adding 10.7ml (0.7 volumes) isopropylalcohol to the elution, mix up.
14) Centrifuge immediately at 15000×g for 30min at 4°C.
15) Remove the supernatant, wash DNA pellet with 5ml of 70% ethanol and centrifuge at 15000× g for 10 min.
16) Remove the 70% ethanol, air-dry the pellet for 10 min, and dissolve the DNA in TE solution.
EXAMPLE 3
[0014] Transfer the recombinant vector-TNK-TPK acquired in the example 2 into host cell HT1080, obtain the required cell lines after screening, and express in vitro, confirms site-directed integration and high efficient expression.
1 Material:
1.1 Cell: HT1080.
Culture medium: high sugar DMEM+10%FBS(HT1080) EMEM+10%FBS.
1.2 Electroporation apparatus: purchased from Bio-Rad Company.
2.Methods
1) Culture the cells in a 75cm 2 culture bottle to 70%∼80% confluence.
2) Harvest the cells and wash with HeBs Buffer for two times, then account the cells.
3) Centrifuge at 15000rpm at 4°C for 10 min.
4) Resuspend the cells with a reasonable volume of HeBS Buffer to make the cell density to 106∼107/ml.
5) Add 0.8 ml suspension (about 10µg DNA vector) to an 0.4cm electroporation cuvette
6) Electroporate the suspension, parameters: 260V, 550uF for 11∼13 ms.
7) Transfer the shocked cells to a 75cm2 culture flask, add 14ml culture medium with ampicillin/streptomycin, then culture in 37°C, 5%CO2 for 24∼48 hrs.
8) Screen the cell with G418 (final concentration of 300µg/ ml), change medium once every 2-3days, in parallel, normal cells is used as control.
9) Calculate the survival clone number in transfected cell after normal cells completely die in 7-10 days, and then add G418 (150µg/ml) to maintain it.
10) Continue to screen the transfected cells with GCV (the ultimate concentration of 500ng/ml)
11) Most cells die in 7-10 days, add GCV 250µg/ml to remain living cells or withdraw GCV, culture the cell up to 70%∼80% confluence, and determine its expressing activity of the transfected gene.
3.RESULTS
The TNK-TPA gene with the vector were transferred into HT1080 cells by electroporation and the positive cell lines were obtained after positive and negative screening (Collection NO. CCTCC: C200006), and confirmed to be site-directed insertion by FISH (Fig. 3) .The results of activity assay of TNK-TPA refer to Table 1.
Expression activities are: negative control HT1080 cells is 0 U/106 cells/ 24 hrs. the positive HT1080 cells is 408 U/106/24 hrs after transfer and 407 U/106 cells/ 24 hrs after 95 days. The expression of TNK-TPA is of highly stability. In addition. expressed protein of TNK-TPA is proved by Western Blotting and amino acid sequencing (Fig. 4).
| The results of activity assay of TNK-TPA for positive HT1080 cells(ug/106 cells/24hrs) | |||
| The days of transfer | T1* | The days of transfer | T15* |
| 33 | 408 | 54 | 188 |
| 37 | 396 | 58 | 204 |
| 60 | 411 | 95 | 114 |
| 68 | 430 | ||
| 74 | 430 | ||
| 88 | 441 | ||
| 90 | 440.9 | ||
| 95 | 407 | ||
| *T1, T15 are two positive cell lines expressing TNK-TPA |
4 The technology of TNK-TPA protein preparation
1)Culture TNK-TPA/ HT1080 cell line with DMEM medium till 80% confluence, and continue to culture them with serum-free, collect the supernatant after 24 hrs which containing expressed TNK-TPA;
2) Filtrate the supernatant with 0.22 µm filter membrane.
3) Concentrate the filtrate 7 times with 10KD ultrafiltration membrane.
4) Pass through PROSEP-LYSINEII chromatography media column.
5) Wash out the nonspecific protein absorbed on the column with 5× column volume of 0.05M PB buffer (pH 7.0).
6) Elute TNK-TPA protein bound to the column with 0.2M Arginine / 0.05M PB (PH7.5);
7) Desalt protein solution with 3KD ultrafiltration membrane, lyophilize it into powder dosage
(1) Using a DNA sequence from the short arms of human D,G group chromosomes, or its homologous sequence as leading sequence for gene targeting to construct a human source gene vector, in which no important physiological function gene is found.
(2)The target gene TNK-TPA is ligated into the human source vector above with routine method, and the recombinant of the vector and TNK-TPA is obtained.
(3)TNK-TPA gene is targeted to nucleolus organizing region of human D,G group chromosomes in host cell HT1080 by the above recombinant and the cell line attained by screening.